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UBC Theses and Dissertations
Investigating early land plant cuticular waxes using physocomitrella patens as a model species Kurschner, Lewis Miles
Cuticular wax research in Arabidopsis thaliana and other important model plant species has led to the elucidation of biochemical pathways for aliphatic wax synthesis in flowering plants, however, we still lack a critical understanding of how these important pathways developed in early land plants. Physcomitrella patens is a model moss species that is used in laboratory research and is believed to live in habitats similar to those found roughly 500 million years ago. Thus, the cuticle present in Physcomitrella can be used to explore the nature of the cuticles from early land plants. The goal of this thesis was to assess whether the proteins involved in production of wax components for the Physcomitrella cuticle are similar to those that produce the well-studied Arabidopsis cuticle. Six hypothesized cuticular wax genes were discovered in Physcomitrella based on sequence similarity to homologs known to be involved in wax biosynthesis in Arabidopsis. PpCERA, PpCERB, PpCERC, and PpCERD were discovered to have close sequence similarity to AtCER1 and AtCER3, while PpFAR1 and PpFAR2 displayed sequence similarity to AtCER4. Knock-out mutants were generated for each of the Physcomitrella candidate genes, except for Ppfar1, and the ensuing cuticular waxes were analyzed via gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectroscopy (GC-MS). A complementation experiment with Physcomitrella genes in knock-out mutants of the homologs in Arabidopsis was performed to determine if the moss genes could complement the wax-deficient phenotype. The Physcomitrella genes were also expressed in yeast to investigate their biological function, but these studies were discontinued due to technical difficulties. Physcomitrella mutants demonstrated that PpFAR2 participates in the production of primary alcohols and functions in the primary alcohol pathway. PpCERA, PpCERB, PpCERC, and PpCERD participate in the production aldehydes and alkanes. When PpCERC and PpCERD were expressed Arabidopsis alkane pathway mutants, Atcer1-4 and Atcer3-6, the wax deficient phenotype was complemented, and alkane pathway functionality was restored. Expression of PpCERC and PpCERD in Atcer4-3 failed to restore the primary alcohol pathway, suggesting that these enzymes may function in the alkane, not primary alcohol, pathway in Physcomitrella.
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